Serum exo-miR584-5p mediates drug resistance in diffuse large B-cell lymphoma by upregulating the drug metabolism-CYP450 pathway, promoting high P450 expression, and remodling its intracellular localization.

Abstract Background Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. Although standard first-line targeted combination therapy cures about 60% to 70% of patients, 30% to 40% still progress to relapsed or refractory (R/R) DLBCL. Exploring the molecular mechanisms underlying chemotherapy resistance in DLBCL, developing new drugs, and devising innovative treatment strategies are crucial for improving patient outcomes and enhancing cure rates. Exosomes, also known as extracellular vesicles (EVs), their unique membrane structure makes them “delivery personnel” that can transport RNA, proteins, and other macromolecules between cells. Exosomal miRNA derived from serum may mediate drug resistance in DLBCL. MethodsClinical data and serum samples were collected from 40 patients diagnosed with DLBCL between March 2021 and November 2024, including 15 patients who achieved a complete response (CR) after R-CHOP-based treatment regimen as the CR group, and 25 patients with relapsed or refractory (R/R) DLBCL as the R/R group.Serum exosomes were extractedby Total Exosome Isolation Kit from 40 samples, and identified by Transmission electron microscopy method（TEM）, Nanoparticle size-potential analyzer method（NTA）, Nano Flow Cytometry method (NFCM), and/or Western blotting (WB).High-throughput sequencingwas used todetect exo-miRNAs in 5 samples from the CR group and the R/R group, respectively, and differentially expressed exo-miRNAs between the two groups were identified. TargetScan, miRDB, and Mahybrid Gene Wibe were utilized for target gene prediction. Biological function analysis, including pathway, GO, KEGG enrichment analyses, and String analysis, was used to speculate the signal transduction pathway in R/R DLBCL.Validationwas involved in 20 cases from the CR group and 10 from the R/R group. The most significantly differentially expressed serum exo-miR-584-5p was isolated, and its relative expression level was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to analyze clinical correlations. Formalin-fixed paraffin-embedded (FFPE) lymphoma tissues from CR and R/R groups were used to detect the expression of cytochrome P450 (CYP) enzymes by immunohistochemistry (IHC), followed by analysis of exo-miR-584-5p, P450, and clinical correlations.Data analysis was performed using SPSS 26.0 and R 4.2. Poisson distribution, Fisher's exact test, and the likelihood ratio test were used to identify differential expressions. Receiver operating characteristic (ROC) curves evaluated diagnostic efficacy, and Kaplan-Meier (K-M) survival curves assessed prognosis. ResultsA total of 79 exo-miRNAs associated with the R/R group were identified, and 11/79 miRNAs were significantly differently expressed among them; miR-584-5p is one of most significantly differently expressed in up-regulated genes. These target genes are primarily located within intracellular and endomembrane systems, participating in key biological processes through drug metabolism and intracellular signaling pathways. The most prominent pathway is drug metabolism via CYP, with miR-584-5p targeting CYP genes in this pathway.The serum exo-miR-584-5p level in the R/R group was higher than in the CR group with a significant difference (p < 0.05), and the area under the ROC curve (AUC) was 0.84 in the research. Exo-miR-584-5p expression was positively correlated with serum lactate dehydrogenase (LDH), and high Exo-miR-584-5p levels predicted a shorter recurrence-free survival (RFS) in the study.CYP450 expression in the R/R lymphoma cell nuclear membranes is stronger than that in the CR group, and in the CR group that is in the endoplasmic reticulum or mitochondrial inner membrane.Exo-miR-584-5p promotes immunochemotherapy resistance by remodling in the subcellular localization of CYP450. The regulation of the CYP450 pathway is primarily centered on cytochrome P450 oxidoreductase (POR), which interacts with endoplasmic reticulum membrane protein complex 2 (EMC2). Conclusions Serum exo-miR-584-5p influences drug resistance in DLBCL via the CYP450 signaling network pathway, by promoting high P450 expression and remodling its intracellular localization, is likely to function as a novel liquid biopsy marker and an independent predictor for R/R DLBCL, and may provide a new target in the future. Keywords: Diffuse large B-cell lymphoma (DLBCL); Drug resistance; Exosomes (Exo); miR-584-5p; Cytochrome P450 (CYP) enzyme